The objective of this study is to determine the biochemical mechanisms that regulate catabolism of the branched chain amino acids (BCAA), leucine, isoleucine and valine. Emphasis will be on determining the metabolic consequences of compartmentation and the role of transport in regulation. The first two steps in the catabolism of these three indispensable amino acids are transamination followed by oxidative decarboxylation of the transamination products, the branched chain alpha-keto acids (BCKA). The mitochondrial enzyme catalyzing the second step, the branched chain alpha-keto acid dehydrogenase, is the first rate-controlling step in BCAA catabolism in vivo, and genetic impairment of this enzyme causes a group of BCKA acidemias collectively known as Maple Syrup Urine Disease. Abnormalities in plasma BCAA also occur in a number of clinical states, and research effort has been directed towards possible clinical uses for BCAA and BCKA. We have established the identify of a mitochondrial transport system for BCKA in mitochondria isolated from rat heart. Thus, our first goal is to understand BCKA transport at a molecular level. The kinetic pattern for BCKA uptake will be determined in rat heart mitochondria. Inhibitor and substrate specificities of the BCKA and pyruvate transporters will be compared, and transporter activity will be defined in tissues that metabolize BCAA. Our aim is development of a kinetic model for the mitochondrial monocarboxylate:H+ cotransporters. Experiments will be designed to purify and reconstitute transporter activity in phospholipid vesicles followed by molecular characterization of the purified proteins. Our second goal is to understand BCAA metabolism. Emphasis will be placed on how the subcellular distribution of BCAA aminotransferase and intramitochondrial location of BCKA dehydrogenase regulate BCAA catabolism. The role of transport and intramitochondrial pH will be examined. Using 31P NMR spectroscopy, characterization of the mitochondrial spectra will provide a basis for determining mitochondrial pH in intact cells. BCAA aminotransferase activity will be assayed in subcellular fractions from rat tissues and activity quantitated relative to a mitochondrial marker enzyme. Preliminary data indicate this enzyme is solely mitochondrial in rat heart. The mitochondrial enzyme will be purified from rat heart, antibodies will be raised, and they will be used to quantitate BCAA aminotransferase antigen in rat tissues. Finally, using isolated mitochondria, an in vitro for BCAA catabolism will be developed, and used to test our hypotheses.